Strains of Saccharaothrix, process for producing pravastain using the strains and isolation process of (HMG)-CoA reductase

ABSTRACT

The present invention provides two new microorganism strains of  Saccharothrix , designated as YS-44442 and YS-45494, a process of producing pravastatin using the strains, and an improved process for isolation of (HMG)-CoA reductase inhibitors.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 10/085,871filed on Feb. 27, 2002 now U.S. Pat. No. 6,716,615.

REFERENCE TO SEQUENCE LISTING

This application incorporates by reference to the computer readable formof the Sequence Listing contained in U.S. application Ser. No.10/085,871 filed Feb. 27, 2002, and titled “Strains of SaccharaothrixProcess for Producing Pravastin Using the Strains and Isolation Processof (HMG)-COA Reductase” (now U.S. Pat. No. 6,716,615).

FIELD OF THE INVENTION

The present invention relates to two new microorganism strains ofSaccharothrix, designated as YS-44442 and YS-45494, a process ofproducing pravastatin using the strains, and an improved process forisolation of (HMG)-CoA reductase inhibitors.

BACKGROUND OF THE INVENTION

It has been recognized that an elevated blood cholesterol level is oneof the major risk factors to atherosclerotic diseases, specifically tocoronary heart diseases. The monitor for the cholesterol biosynthesis isvery helpful to control the diseases. 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase is the rate-limiting enzyme in the cholesterolbiosynthesis. By inhibiting the activity of (HMG)-CoA reductase, bloodcholesterol levels in the bodies can be effectively reduced.

A number of (HMG)-CoA reductase inhibitors have been discovered, such aspravastatin, compactin, lovastatin. They have the following formula inthe lactone form and may exist in other forms such as the acid form orand the salts and esters thereof.

These (HMG)-CoA reductase inhibitors are very effective in loweringblood cholesterol level in most animals including human. Pravastatin iseven more active than compactin and lovastatin, and has been applied inthe treatment of hypercholesterolemia (Nara, F et al. Curr. Genet. 23:28–32 (1993)).

Pravastatin is a 3_-hydroxy derivative of compactin. It has beenreported that pravastatin is produced by converting compactin throughmicrobial hydroxylation using various genera of fungi and bacteria, suchas Streptomyces roseochrornogenus (U.S. Pat. No. 4,346,277) andActinomadura sp. (Peng, M. et al., J. Antibiotics, Dec: 1032–1035(1997), and Peng Y. and A. L. Demain, J. Mol. Cataly. B: Enzymatic 10:151–156 (2000)). However, these fungi or bacteria do not tolerate a highamount of compactin added in the fungal or bacterial fermentation broth,likely due to the anti-fungal activity of compactin, and thus exhibitlow productivity of pravastatin. Therefore, there is a need to find anew microorganism which is tolerable to a higher amount of compactin andhas effective conversion activity.

In general, the isolation of (HMG)-CoA reductase inhibitors from afermentation broth is conducted by serious procedures of extraction,chromatography, lactonization and crystallization process. EP 0 877089A1 discloses a (HMG)-CoA reductase inhibitor preparation process,wherein a fermentation broth containing the inhibitor (e.g., lovastatin)is basified prior to filtration to remove the cells and then thefiltrate was loaded through a column. The eluate was extracted withtoluene and subsequently the lactonization is conducted to produce theinhibitor.

When using chromatography, however, a large column and a great volume offermentation broth containing (HMG)-CoA reductase inhibitors are usuallyneeded to obtain a desired yield of the inhibitors, thereby increasingthe difficulty in handling the purification process of the inhibitors.Furthermore, a lactonization reaction usually needs much energy andtime. Therefore, there is a need to seek an improved process ofobtaining a (HMG)-CoA reductase inhibitor in a good yield and puritywithout proceeding a lactonization reaction and using a chromatography,and so as to reduce the cost.

SUMMARY OF THE INVENTION

One object of the invention is to provide two new microorganism strainsof Saccharothrix capable of producing pravastatin designated as YS-44442and YS-45494 as well as their mutants.

Another object of the invention is to provide a process of producingpravastatin by using the microorganisms of the invention. In particular,the process of producing pravastatin comprises the steps of (a)cultivating YS-44442 or YS-45494 at a suitable condition to generate afermentation broth; (b) feeding compactin into the broth; (c) fermentingthe broth for a period of time to convert the compactin to pravastatin;(d) isolating the pravastatin from the broth.

Still another object of the invention is to provide a process ofisolating a (HMG)-CoA reductase inhibitor comprising the steps of (1)adding an ammonium sulfate into a first solution containing the(HMG)-CoA reductase inhibitor to produce a precipitation; (2) isolatingthe precipitation; (3) dissolving the precipitation with a polar solventto produce a second solution; (4) adjusting the pH of the secondsolution to about 4 to about 6; and (5) extracting the second solutionwith an water immiscible solvent to isolate the (HMG)-CoA reductaseinhibitor. The preparation process further comprises a step of reactingthe isolated (HMG)-CoA reductase inhibitor with an organic or inorganiccation source to generate a salt form of the inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a 16s rDNA phylogenetic distance tree comparing YS-44442 andYS-45494 of the invention with related actinomycetes generated with theARB software package (denoted as Tree 0.1 version).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be more fully understood and furtheradvantages will become apparent when reference is made to the followingdescription of the invention.

New Microorganism Strains

One object of the invention is to provide two new microorganism strainsfor producing pravastatin in a good yield. The inventors surprisinglyobtained two novel microorganism strains, YS-44442 and YS-45494, throughthe isolation of actinomycetes from soil samples, showing a hightolerance to compactin and a high productivity of pravastatin. The twostrains were deposited with the China Center for Type Culture Collection(CCTCC) under the accession number of M202001 and M202002, respectively,on 8 Jan. 2002.

YS-44442 and YS-45494 can be isolated from soils. A soil sample issuspended in a sterile phosphate buffer and spread, after a 10-foldserial dilution, on an agar plate containing an isolation medium. Thecolonies growing on the plates can be preliminarily screened based onthe morphological characteristics of actinomycetes under a microscopyexamination. A candidate strain is then cultivated to give afermentation broth and fed with compactin to produce pravastatin. Theamount of pravastatin thus produced in the broth is determined. Then,YS-44442 and YS-45494 are selected for their high productivities ofpravastatin.

The medium of the agar plate is well known in this art, preferablycontaining yeast extract, malt extract, dextrose and agar, and morepreferably ISP 2 medium. In certain embodiments, the isolation reagentcontained in the medium preferably contains antibiotics which can beselected from nystatin, cycloheximide, penicillin G, polymyxin B andgentamycin. The morphological characteristics of the genus,Actinomycetes, have been well described in conventional literatures ortext books, such as Bergey's Manual of Systematic Bacteriology [Cross,T., Bergey's Manual of Systematic Bacteriology, S. T. Williams, Sharpe,M. E., Holt, J. G., Editor (1989) Williams & Wilkins: Baltimore. p.2586–2615]. In addition, the technologies of cultivation or fermentationof such microorganisms are well known in the art.

The compactin to be fed into a broth is preferably provided by amicroorganism (e.g., fungus or bacterium) producing compactin or thecell extract thereof, or a solution comprising compactin. Theconcentration of compactin fed into a broth is calculated based on thebroth without microbe and only the feeding compactin. Pravastatinproduced in a fermentation broth can be identified by conventionaltechnologies, such as HPLC, and the amount of pravastatin can bedetermined by the retention time in comparison with a standardpreparation. For example, the yield of pravastatin produced can bedetermined by Formula (I). In addition, the conversion efficiency ofcompactin to pravastatin can be calculated by Formula (II) and theepi-pravastatin ratio( Epi %) of the total pravastatin produced iscalculated by the formula (III).Pravastatin yield=(standard concentration/standard Area)*samplearea  (I)Molar conversion efficiency(ME %)=(Number of moles of pravastatinproduced/Number of moles of compactin converted)*100  (II)Epi-pravastatin ratio(Epi %)=(amount of Epi-pravastatin produced/(amountof Epi-pravastatin produced+amount of pravastatin produced))*100%  (III)

Epi-pravastatin is a 6_-hydroxylation form of compactin which does nothave a desired therapeutic efficacy and even has a negative effect inclinical therapies. Therefore, a low epi-pravastatin ratio is preferred.In addition, the term “compactin tolerance” used herein means a finalconcentration of compactin fed into a microbial fermentation brothwherein the concentration is accepted by the microorganism, i.e., themicroorganism can normally grow in a medium containing such amount ofcompactin.

The YS-44442 obtained as described above has the following properties:the colonies (7 days) on plates are pale white without sporulation; theculture in SCY agar slant are pale white with few spores; the conversionefficiency of compactin to pravastatin (ME %) is about 70 to 75%; theepi-pravastatin ratio is 6.5 to 8%; the production of pravastatin is 1.0to 1.5 g/L by volume of the fermentation broth; and the compactintolerance is 1.5 to 2.0 g/L. More detailed characteristics of YS-44442are described in the following examples.

The YS-45494 obtained as described above has the following properties:the colonies (7 days) on plates are white with sporulation; the culturein SCY agar slant produces white spores and yellow pigments; theconversion efficiency of compactin to pravastatin (ME %) is about 48 to50%; the epi-pravastatin ratio is 1.8 to 3.0%; the production ofpravastatin is 1.0 to 1.5 g/L by volume of the fermentation broth; andthe compactin tolerance reaches 1.5 to 2.0 g/L. More detailedcharacteristics of YS-45494 are described in the following examples.

Mutants derived from YS-44442 and YS-45494 capable of producingpravastatin are also included in the invention. The mutants can beobtained by applying a conventional mutation-inducing technique to theparent YS-44442 and YS-45494. For example, irradiation of themicroorganisms with gamma rays or ultraviolet light, or treated with amutagen, such as EMS (ethylmethane sulfonate), NTG(N-methyl-N-nitro-N′-nitrosoguanidine, NQO (4-nitroquinoline N-oxide),DES (diethylsulfate), DEB (diepoxybutane) and NMU(N-methyl-N-nitrosourea) are suitable methods of inducing mutation. Toobtain a mutant, the parent strain YS-44442 and YS-45494 may be treatedwith UV irradiation for 30, 60, 120, 240 or 480 seconds, or with cobalt60 gamma irradiation in an amount of 0.5, 1, 2, 3 or 4 Kgy. A desirablemutant strain capable of producing pravastatin in good yield can beselected by the method as described above. Persons skilled in the artcan obtain such mutants from the parent YS-44442 and YS-45494 accordingto the mutagenesis technologies and screening method conventional to theart. More detailed procedures of the mutagenesis procedures aredescribed in the following examples.

Process for Producing Pravastatin Using the Strains of the Invention

YS-44442 and YS-45494 can be used to produce pravastatin. Accordingly,one object of the invention is to provide a process of producingpravastatin of using YS-44442 and YS-45494. In particular, the processcomprises the steps of (a) cultivating YS-44442 or YS-45494 in asuitable condition to generate a fermentation broth; (b) feedingcompactin into the broth; (c) fermenting the broth for a period of timeto convert the compactin to the pravastatin; (d) isolating thepravastatin from the broth.

The technologies of cultivating such microorganisms and those ofisolating pravastatin from a fermentation broth are well known in theart. Persons skilled in the art can accomplish the production process ofpravastatin using YS-44442 and YS-45494 of the invention in combinationwith any fermentation and isolation technologies (e.g., HPLC) known inthe art. A particular example is illustrated later.

In one embodiment of the invention, the fermentation broth of Step (a)is incubated for less than 2 days and preferably for about 18 hours. Thefermentation broth of Step (a) is preferably derived from a seed cultureof the microorganism which is cultivated under a suitable condition forabout 18 to 48 hrs. A seed medium for cultivating the seed culture cancontain glucose, peptone, soy protein and mineral sources. Afermentation medium can further contain corn steep powder (C.S.P.).

The compactin to be added in the broth is preferably provided by afungus or bacterium capable of producing compactin or the cell extractthereof, or a solution comprising compactin. The concentration ofcompactin fed in a broth is calculated based on the broth withoutmicrobe and only the feeding compactin. In a preferred embodiment of theinvention, the compactin is fed into the broth in a final concentrationof higher than 1.0 g/L and more preferably in a range of 1.5 to 2.0 g/L.

In another embodiment of the invention, the period of time of Step (c)to convert the compactin to the pravastatin is less than 5 days,preferably less than 3 days and more preferably less than 24 hours.

In one preferred embodiment of the invention, the pravastatin isolatedfrom Step (d) yields more than 0.5 g/L, preferably more than 1.0 g/L andmost preferably about 1.5 g/L. In one preferred embodiment of theinvention, the epi-pravastatin ratio (EP %) of the total pravastatinproduced from Step (d) is about 1.8 to 8%. In one preferred embodimentof the invention, the conversion efficiency (ME %) of compactin topravastatin is higher than 40%, more preferably higher than 50%, stillmore preferably higher than 70%. The pravastatin produced form the aboveprocess can be isolated by any conventional technologies in the art,such as HPLC, and further extracted or crystallized.

Process for Isolating Pravastatin Without Using Chromatography

The inventors developed an improved process of isolating a (HMG)-CoAreductase inhibitor in a good yield and purity without using achromatography and proceeding a lactonization reaction.

Accordingly, one object of the invention is to provide a process ofisolating a (HMG)-CoA reductase inhibitor which comprises the steps of(1) adding an ammonium sulfate into a first solution containingthe(HMG)-CoA reductase inhibitor to produce a precipitation; (2)isolating the precipitation; (3) dissolving the precipitation with apolar solvent to produce a second solution; (4) adjusting the pH of thesecond solution to about pH 4 to about PH 6; and (5) extracting thesecond solution with an water immiscible solvent to isolate the(HMG)-CoA reductase inhibitor.

In a preferred embodiment of the invention, the (HMG)-CoA reductaseinhibitors is selected from pravastatin, compactin and lovastatin, andmore preferably pravastatin.

The first solution of Step (1) of the present isolation process can beany solution containing a (HMG)-CoA reductase inhibitor to be isolated.In a preferred embodiment of the invention, the first solution of Step(1) of the present isolation process is a microbial fermentation brothwhich can be derived from any microorganism capable of producing the(HMG)-CoA reductase inhibitor. The microorganism is preferably selectedfrom Streptomyces roseochrornogenus (U.S. Pat. No. 4,346,277),Actinomadura sp. (Peng, M. et al. supra and Peng Y. and A. L. Demain,supra). Aspergillus, Monascus, Penicillium, Paecilomyces, Hypomyces,Phoma, Pleurotus, Doratmyces, Eupenicillium, Gymnoaxus, Trichoderma (EP0 877 089 A1), more preferably YS-44442 and YS-45494 of the invention,and their mutants described herein.

The ammonium sulfate of Step (1) is preferably added into the firstsolution in an amount of 30 to 60% (w/v) of the first solution. Morepreferably, the ammonium sulfate is added to be saturated in the firstsolution.

In Step (1) of the present isolation process, the precipitation can beisolated by any method known in this art, such as filtration,centrifugation or decantation, and a membrane filtration is preferred.

In a preferred embodiment of the invention, the pH of the secondsolution of Step (4) is adjusted with an inorganic acid, preferably HCl.Persons skilled in the art can select a proper acid according toconventional technologies and knowledge to adjust the pH of the secondsolution.

In Step (5) of the present isolation process, the technology used toextract the second solution with an water immiscible solvent is wellknown in this art. Persons skilled in the art can select and use aproper water immiscible solvent to successfully extract the (HMG)-CoAreductase inhibitor from the second solution. Preferably, the waterimmiscible solvent is an organic solvent which can be selected fromethyl acetate, acetone, toluene, dicholoromethane and isopropyl acetate,and more preferably ethyl acetate. The amount of the organic solvent tobe added is dependent on the concentration of the (HMG)-CoA reductaseinhibitor contained in the second solution. The time period ofextraction is preferably more than 5 minutes, most preferably 5 to 30minutes.

After extraction with an organic solvent, the organic solvent layer iscollected, and then dried and de-colorized using the conventionaltechnologies (such as anhydrous magnesium sulfate and activated carbon)to obtain an isolated (HMG)-CoA reductase inhibitor.

In one embodiment of the invention, the present isolation processfurther comprises a step of reacting the isolated (HMG)-CoA reductaseinhibitor with an organic or inorganic cation source, preferably sodium,to generate a salt form of the inhibitor. The sodium source ispreferably selected from NaOH, Na₂CO₃, sodium acetate (anhydrous) andsodium-2-ethyl hexanoate. The amount of cation source added and thereaction duration is dependent of the concentration of the (HMG)-CoAreductase inhibitor. Persons skilled in the art can select a properspecies and amount of the cation source to obtain the salt form of theinhibitor. Preferably the cation source is added in an concentration of0.2 to 5.0 M with stirring for 0.5 to 2.0 hours.

Without any intention to limit it in any manner, the present inventionwill be further illustrated by the following examples.

EXAMPLES Example 1

A. Isolation of Strains YS-44442 and YS-45494 from Soil Samples

Soil samples used for the isolation of actinomycetes were collected fromrandom sites. The soil samples were kept at room temperature until theydried to constant weight. One gram of an air-dried soil sample wassuspended in 10 ml of sterile 5 mM phosphate buffer (pH 7.0) and stirredfor 1 min in a super mixer. To germinate actinomycetes spores, thesuspension of the soil sample was heated at 35° C. to 50° C. for 10 minin a shaking incubator. After a 10-fold serial dilution, the suspensionwas spread on agar plates containing suitable isolation medium. Coloniesoccurring on the plates were counted with naked eyes and examined undera light microscope for the morphological characteristics ofactinomycetes. 400 strains showing positive morphologicalcharacteristics were selected from 3000 unidentified colonies. Due to ahigh conversion efficiency (>50%) from compactin to pravastatin and lowepi-pravastatin content (<10%), strains YS-44442 and YS-45494 wereselected among the 400 positive strains. The isolation conditions ofYS-44442 and YS-45494 are described in Table 1.

TABLE 1 Isolation conditions of strains YS-44442 and YS-45494 strain no.YS-44442 YS-45494 Soil source Taipei, Taiwan Australian desert Soil pH5.4 9.6 pretreatment moist heat at 50° C. moist heat at 40° C. for 10min for 10 min Selective Nystatin 50_g/ml Nystatin 50_g/ml antibioticsCycloheximide 50_g/ml Cycloheximide 50_g/ml Pen G 1_g/ml Gentamycin1_g/ml Polymyxin B 5_g/mlB. Characteristics of Strains YS-44442 and YS-45494

Strain YS-44442 or YS-45494 was maintained on slants of yeastextract-malt extract agar (ISP-2) [Shirling, E. B. and Gottlieb, D.,International Journal of Systematic Bacteriology, 16(3): 313–340 (1966)]and grew at 27° C. Inoculum for physiological tests and preparation ofbiomass for DNA extraction were prepared by growing in tryptone-yeastextract broth (ISP-1) [Shirling and Gottlieb supra].

(1) Morphology and Pigment Production

Morphology characterizations were performed as described by Shirling &Gottlieb supra. Culture morphology was examined by light microscopyusing a Nikon Optiphot-2 microscope equipped with 45× and 100×super-long working distance objectives. Color determination of theaerial mycelium was made by observation of the mature, sporulatingaerial surface growth. Color determination of the substrate mycelium anddiffuisible soluble pigments, other than melanins, was made byobservation of the reverse side. Substrate mycelium coloration wasassigned to one of the following color groups: (1) yellow-brown; (2)yellow-brown+red (or orange); (3) yellow-brown+blue (or violet); (3)yellow-brown+green. Pigment and color determinations were defined basedon comparison to the Tektronix© RGB color sampler (Xerox, Inc.).Morphological characterizations were carried out on yeast extract-maltextract agar (ISP-2), oatmeal agar (ISP-3), inorganic salts-starch agar(ISP-4), and glycerol-asparagine agar (ISP-5) [Shirling and Gottliebsupra].

With respect to the results of YS-44442, on ISP-2 and ISP-4, short,flexuous (open) hooked or (short) spiraled chains of 5 to 10 spores wereobserved. The spores were smooth and spherical and were appropriately 2to 3 times the hyphal diameter. Pseudosporangia were present on ISP-2and ISP-4 but were more abundant on ISP-4. Aerial mass coloration waswhite (R100 G100 B100) on ISP-2 and ISP-3. No aerial mycelia wereproduced on either ISP-4 or ISP-5 (see Table 2). In addition, thecoloration of the substrate mycelium of YS-44442 was brown (R85 G70 B40,color 2) on ISP-2, ISP-3 and ISP-4, and yellow-brown (R100 G100 B70,color 1) on ISP-5 (see Table 2). Pigments did not change in response topH changes following addition of approximately 100 μl of 0.05 N NaOH and0.05 N HCl. Furthermore, no soluble pigments were produced by YS-44442(see Table 2).

As to the result of YS-45494, aerial mass coloration of the aerialmycelium was white (R100 G100 B100) on ISP 2, 3, 4, and 5. On ISP-2, theaerial mycelium was long, straight, and branched. On ISP-3, the aerialmycelium coalesced with some swelling evident at the tips of the hyphalvegetative growth. Upon aging, strain A5 fragmented into cocco-vassilaryelements on ISP-5. The coloration of substrate mycelium was orange-brown(R100 G85 B40, group 2) on ISP-2, red-brown (R70 G55 B40, group 2) onISP-3, light yellow-brown (R100 G100 B85, color group 1) on ISP-4, andlight brown (R100 G100 B70, group 1) on ISP-5 (see Table 2). Pigmentsdid not change in response to pH changes following addition ofapproximately 100 μl of 0.05 N NaOH and 0.05 N HCl. In addition, solublepigments were observed on ISP-3 and ISP-5. Coloration was brown (R70 G55B40) on ISP-3 and yellow (R100 G100 B85) on ISP-5 (see Table 2). Neithersoluble colors changed in response to pH changes following addition ofapproximately 100 μl of 0.05 N NaOH and 0.05 N HCl.

The morphological characteristics observed of YS-44442 and YS-45494 aresummarized in Table 2.

TABLE 2 Colony morphology of strains YS-44442 and YS-45494 YS-44442 2White Brown (group 2) None 3 White Brown (group 2) None 4 None Brown(group 2) None 5 None yellow-brown (group 1) None YS-45494 ISP Aerialmass Soluble Medium color Substrate mycelium color colors 2 WhiteOrange-brown (group 2) None 3 White Red-brown (group 2) Brown 4 WhiteLight yellow-brown None (group 1) 5 White Light yellow-brown Yellow(group 1)(2) Physiological Tests

Melanin production and sole carbon source utilization were determined asdescribed by Shirling & Gottlieb supra. Melanin production was evaluatedon peptone-yeast extract iron agar (ISP-6) and tyrosine agar (ISP-7)[Shirling & Gottlieb supra]. Sole carbon source utilization wasevaluated on basal mineral salts agar (ISP-9) amended with the followingto 1% (w/v) concentration: D-glucose (positive control), i-arabinose,sucrose, D-xylose, i-inositol, D-mannitol, D-fructose, rhamnose,raffinose, and cellulose [Shirling & Gottlieb supra]. The negativecontrol consisted of un-amended basal mineral salts agar. Results werescored as described by Shirling & Gottlieb [Shirling & Gottlieb supra].

The results of the general growth characteristics of YS-44442 andYS-45494 are given in Table 3.

TABLE 3 General growth characteristics of YS-44442 and YS-45494 StrainYS-44442 YS-45494 ISP medium Growth Growth 2 Moderate Moderate 3Moderate Moderate 4 Fair Fair 5 Fair Fair 6 Fair Fair 7 Fair Fair

With respect to the carbohydrate utilization patterns of YS-44442,strongly positive utilization was observed on L-arabinose, sucrose,D-fructose, d-fructose and rhamnose. Doubtfuil utilization was observedon cellulose and negative utilization was observed on I-inositol andraffinose. No melanoid pigments were produced by the culture.

As to the carbohydrate utilization patterns of YS-45494, stronglypositive utilization was observed on L-arabinose, sucrose, andD-fructose. Positive utilization was observed on rhamnose and raffinose.Doubtful utilization was observed on D-xylose, I-inositol, D-mannitol,and cellulose. No melanoid pigments were produced by the culture.

The results of the carbohydrate utilization patterns of YS-44442 andYS-45494 are given in Table 4.

TABLE 4 Carbohydrate utilization patterns Strain YS-44442 YS-45494 Day10 13 16 10 13 16 none − − − − − − D-glucose ++ ++ ++ ++ ++ ++T-arabinose ++ ++ ++ + + ++ sucrose ++ ++ ++ ++ ++ ++ D-xylose ++ ++ ++− +/− +/− i-inositol − − − +/− +/− +/− D-mannitol ++ ++ ++ +/− +/− +/−D-fructose ++ ++ ++ + + ++ rhamnose ++ ++ ++ + + + raffinose − − − + + +cellulose − − +/− +/− +/− +/− ++ = strongly positive utilization + =positive utilization +/− = utilization doubtful − = utilization negative(3) 16s rDNA Sequence DeterminationDNA Extraction

Biomass for DNA extraction was prepared by growing in 50 ml oftryptone-yeast extract broth (ISP- 1) for 10 days, with continuousshaking at 250 rpm on an orbital shaker (1-inch throw), at 27° C.[Shirling & Gottlieb supra]. The liquid culture was harvested bycentrifugation for 15 minutes at 15,000×g. The biomass was transferredto a sterile mortar and pestle, and gently ground into a paste.Approximately 0.4 ml of TE buffer (10 mM tris: 1 mMethylenedinitrolotetraacetic acid) was added and the buffer and celldebris were transferred to a sterile 2-ml centrifuge tube with 100 μl of10% SDS (sodium dodecyl sulfate) solution. The mixture was incubated ina water bath at 65° C. for 15 minutes. Approximately 0.5 ml of bufferedphenol (pH 8) [Ausubel, F. M., Short protocols in molecular biology, 3rded (1995), New York, Wiley. I v. (various pagings)] was added to thetube and mixed for 10 minutes. The mixture was centrifuged at 14,474×gfor 15 minutes. The aqueous layer was transferred to a sterile 2-mlcentrifuge tube and 0.5 ml of chloroform-isoamyl alcohol (24:1) wasadded. The mixture was centrifuged at 14,474×g for 15 minutes and theaqueous layer was transferred to a sterile 2-ml centrifuge tube.Approximately 50 μl of 5 M NaCl and 1 ml of cold 95% ethanol were addedto the tube, and the DNA was precipitated by holding the mixture −20° C.for 24 hours. The mixture was centrifuged at 14,474×g, for 30 minutes at4° C., and the supernatant removed. Approximately 100 μl of 80% ethanolwas added to the tube followed by centrifugation at 14,474×g for 10minutes. The ethanol was removed and the pellet was dried for 30 minutesand re-suspended in 50 μl of sterile deionized water (18 Mohm). The DNAwas stored at −20° C. until ready for use.

16s rDNA Sequence Determination

PCR amplification of the 16s rDNA sequence was performed on theextracted DNA using GibcoBRL® native Taq polymerase on a Perkin-ElmerGeneAmp® PCR System 2400. Purification of the amplified 16s rDNA wasaccomplished with the Bio-Rad Prep-A-Gene® DNA Purification System.Fluorescent labeled DNA for sequencing was prepared using the ABI Prism®BigDye™ Terminator Cycle Sequencing Ready Reaction Kit. DNA sequence wasdone on an ABI Prism® 310 Genetic Analyzer equipped with a 61 cm×50 μmcapillary using the ABI Prism®POP-6™ Performance Optimized Polymer 6according to manufacturer's instructions. Primers GMG 1 and GMG 10 wereused in 16s rDNA PCR amplification and sequencing (see Table 5). The 16srDNA PCR amplification program is as follows: (1) 94° C. for 7 minutes;(2) 94° C. for 1 minute; (3) 53° C. for 1 minute; (4) 72° C. for 2minutes; (5) 72° C. for 6 minutes; steps 2 to 5 were repeated for 30cycles; and (6) Hold at 4° C.

TABLE 5 Primers used in 16s rDNA PCR amplification and sequence PrimerDirection Sequence (5′

3′) GMG 1   27 Forward GAG TTT GAT CCT GGC TCA G (SEQ ID NO: 3) GMG 21385 Reverse CGG TGT GTR CAA GGC CC (SEQ ID NO: 4) GMG 3 1114 ForwardGCA ACG AGC GCA ACC C (SEQ ID NO: 5) GMG 4  907 Reverse CCG TCA ATT CATTTG AGT TT (SEQ ID NO: 6) GMG 5  803 Forward ATT AGA TAC CCT GGT AG (SEQID NO: 7) GMG 6  536 Forward CAG CMG CCG CGG TAA TWC (SEQ ID NO: 8) GMG7  519 Reverse GWA TTA CCG CGG CKG CTG (SEQ ID NO: 9) GMG 8  357 ForwardTAC GGG AGG CAG CAG (SEQ ID NO: 10) GMG 9  343 Reverse CTG CTG CCT CCCGTA (SEQ ID NO: 12) GMG 10 1525 Reverse AGA AAG GAG GTG ATC CAG CC (SEQID NO: 13)

The 16s rDNA sequences of YS-44442 and YS-45494 comprise the sequencesas shown in SEQ ID NO: 1 and 2, respectively. The sequences were alignedagainst the multiple sequence alignment dataset in the RibosomalDatabase Project (RDP) [Maidak, B. L. et al., Nucleic Acids Res., 28:173–174 (2000)] using the ARB sequence editor (release 8.1). Comparisonsof the 16s rDNA sequences of YS-44442 and YS-45494 to the RDP [Maidak,B. L. el al., supra] shows 99.1% and 99.0% sequence similarity,respectively, to Saccharothrix sp. NRRL B-16133 [Labeda, D. P. and R. M.Kroppenstedt, Int. J Syst. Evol. Microbiol., 50 Pt 1: 331–6 (2000)].(http://www.eme.msu.edu/RDP/html/index.html). In addition, 16s rDNAphylogenetic distance tree, comparing YS-44442 and YS-45494 with relatedactinomycetes (Table 6), generated with the ARB software package(denoted as tree 0.1) is shown in FIG. 1.

TABLE 6 Actinomycetes related to strains YS-44442 and YS-45494 GenBankTree Accession Organism Designation U48842 Nonomuraea Africana Nm.african AB006156 Sebekia benihana strain IFO14309 AB006156 U48977Nonomuraea polychroma Nm. plychrm U48975 Nonomuraea helvata Nm. helvataU28978 Nonomuraea pusilla Nm. pusill2 U48843 Nonomuraea angiospora Nm.angiosp X97892 Nonomuraea salmonea Nm. salmone AB006158Cathayosporangium alboflavum strain AB006158 IFO16009 U48999Streptosporangium vulgare Sts. vulgar X99942 Streptomyces vellosusX99942 X60514 Streptomyces coelicolor Stm. cocli3 X79853 Streptomyceshygroscopicus Stm. hyscop D85121 Streptomyces virginiae strain ATCC Stm.virgi5 13161 Y15502 Streptomyces griseus Y15502 X53194 Saccharopolysporarectivirgula Scp. rectiv X53198 Saccharopolyspora erythraea Scp. erythrAF154128 Saccharopolyspora flava AF154128 AF002818 Saccharopolysporaspinosa Scp. spinos AB020031 Saccharothrix tangerinus strain MK27-AB020031 91F2 AF114804 Saccharothrix aerocolonigenes AF114804 AF114815Saccharothrix texasensis strain NRRL B- AF114815 16107T AF114807Saccharothrix espanaensis AF114807 X76966 Saccharothrix mutabilis ssp.mutabilis Sct. mutabm DSM 43853 X76965 Saccharothrix mutabilis ssp.capreolus Sct. mutabc DSM 40225 M29282 Saccharothrix australiensis Sct.austra AF114803 Saccharothrix australiensis Sct. austr2 AF114812Saccharothrix syringae AF114812 AF114805 Saccharothrix coeruleofuscaAF114805C. Comparison of YS-44442 and YS-45494 with Known Microorganisms

YS-44442 and YS-45494 both exhibit phenotypic characteristics similar tothose described by Bergey's Manual of Systematic Bacteriology [Cross,T., supra] for Saccharothrix aerocolonigenes and Saccharothrixaustraliensis, and those described by Labeda and Lyons [Labeda, D. P.and Lyons, A. J., International Journal of Systematic Bacteriology,39(3): 344–358 (19689)] for Saccharothrix texasensis (Table 7). YS-44442and YS-45494 differ in morphology and carbohydrate utilization patternsfrom published descriptions (Table 8). They also differ in carbohydrateutilization patterns with Saccharothrix aerocolonigenes, Saccharothrixaustraliensis, and Saccharothrix texasensis, (Table 8).

TABLE 7 Comparison of YS-44442 and YS-45494 to the descriptions ofrelated microorganisms described in Cross, T. supra and Labeda and Lyonssupra Saccharothrix Saccharothrix Saccharothrix aerocolonigenesaustraliensis texasensis YS-44442 YS-45494 Aerial Color white white towhite white white Mycelium yellowish-gray Morphology fragmentedfragmented no no description fragmented into coccoid description intococco- elements vassilary elements Spore Morphology no description nodescription no short theyous no description cham description hooked orspiral spore chains Number no description no description no 5–10 sporesno description description cham Spores Morphology* no description nodescription no smooth and no description description spherical and 2– 3Xthe hyphal diameter Substrate Color yellowish to brownish to dark yellowbrown to orange-brown, Mycelium brownish yellowish-gray to brownishyellow-brown red-brown or yellow depending on light yellow- the growthbrown medium depending on the growth medium Morphology branchedfragmented no presence of some swelling into coccoid descriptionPseudosporangia at the elements tips of the hyphal vegetative growthSoluble Color yellowish to brownish brown to none brown or pigmentbrownish reddish yellow brown depending on the growth media *as viewedby light microscopy

TABLE 8 Comparison of YS-44442 and YS-45494 to the carbohydrateutilization pattern of related microorganisms described in Cross, T.supra and Labeda and Lyons supra Utilization of carbon SaccharothrixSaccharothrix Saccharothrix Strain YS- Strain YS- compoundsaerocolonigenes australiensis texasensis 45494 44442 no carbon − − − − −control D-glucose + + + + + L-arabinose + − + + + sucrose + − + + +D-xylose + − + − + I-inositol − − + − − D-mannitol no information noinformation no information − + D-fructose + + variable + + Rhamnose −− + + + Raffinose − − − + − Cellulose − − no information − −

Based on the combined results of phylogenetic and physiologicalanalyses, we believe strains YS-44442 and YS45494 are distinct from oneanother and from the type strains of Saccharothrix aerocolonigenes,Saccharothrix australiensis, and Saccharothrix texasensis and are novelspecies of the genus Saccharothrix. The two strains were deposited withthe China Center for Type Culture Collection (CCTCC) and given theaccession number of M202001 and M202002, respectively, on 8 Jan. 2002.

Example 2

Production of Pravastatin by YS-44442 and YS-45494

(1)Preparation of Compactin Solution to be Added in a Fermentation Broth

0.3 N NaOH solution (500 ml) was warmed and kept at 50 to 60_. The NaOHsolution was added with 40 g compactin and mixed for 2 to 3 hours at 50to 60_. The compactin solution in NaOH was cooled down to roomtemperature and the pH was adjusted to 7.5 with 1N HCl. The volume ofthe compactin solution in NaOH was adjusted to 1000 ml and centrifugedat 1000 rpm for 20 minutes. The supernatant was sterile by filtration.

(2) Production of Pravastatin Using YS-44442 and YS-45494

1 ml of a spore suspension of YS-44442 (or YS-45494) stored inGL(10_glycerol_(—)5_lactose) at −80_was opened in laminar flower andadded into a 300 ml shake flask containing 20 ml of the seed medium (forevery liter: glucose 10 g, peptone 2 g, soy protein 4 g and KH₂PO₄ 1 g,pH 7.0÷0.2). The seed culture was incubated at 27_ for 24 to 48 hrs in arotary shaker at 200 to 220 rpm (growth phase). Afterward, 1.5 to 2 mlof the seed culture was incubated into a 300 ml shake flask containing20 ml of the fermentation medium (per liter: glucose 15 g, peptone 5 g,corn steep powder (C.S.P.) 5 g and soybean meal 4 g, KH₂PO₄ 1 g, pH7.0±0.2). The shake flask was incubated at 27±0.5_ for 18 to 24 hrs in arotary shaker at 200 rpm (growth phase). The fermentation broth wasadded with compactin prepared as above into the fermentation broth at afinal concentration of 1500 to 2000 mg/L and the incubation wascontinued for 16 to 24 hrs.

(3) HPLC Analysis of Pravastatin and Compactin

The fermentation broth (0.2 ml) was added into 1.8 ml deionized water.The mixture was mixed for 1 minute and centrifuged at 3000 rpm for 10minutes. The supernatant was analyzed by HPLC using the followingconditions.

-   Column: Nuclosil₅ C₁₈, 5 μm(4.6*150 mm)-   Mobile phase: A:0.1%(H₃PO₄/CH₃CN/CH₃OH=60/32/22)-   (*gradient) B:CH₃CN-   Inject volume: 10_(—)1-   Flow rate: 1.0 ml/min-   Detection: 240 nm-   Retention time: Epi-pravastatin (pravastatin epimer)_(—)6.25 min-    pravastatin_(—)6.85 min-    compactin_(—)9.85 min

* The mobile phase was flew with the following gradient: Time(min) Flow% A % B 0 1.0 100 0 1.0 1.0 100 0 1.1 1.0 30 70 3.5 1.0 30 70 3.6 1.0100 0 30 1.0 100 0 31 0.0 100 0(4) Calculation and Results

A standard pravastatin or compactin preparation dissolved in alkalinemethanol (100% methanol and 0.1 N NaOH) and diluted with H₂O wereprepared. Pravastatin yield, molar conversion efficiency (ME %) andepi-pravastatin ratio (Epi %) are calculated by Formula (I), (II) and(III) as stated above.

The conversion efficiency of compactin to pravastatin, the pravastatinyield and the epi-pravastatin ratio of YS-44442 and YS-45494 are shownin Table 8.

TABLE 8 Results of Pravastatin production by strains YS-44442 andYS-45494 Pravastatin conversion Epi-pravastatin Strain yield efficiencyratio YS-44442 1.0 to 1.2 g/L 70 to 75% 6.5 to 8% YS-45494 1.0 to 1.5g/L 48 to 50% 1.8 to 3.0%

Example 3

Production of Pravastatin by YS-44442 and YS-45494 with ContinuousFeeding of Compactin

Compactin solution to be added in a fermentation broth was prepared asdescribed in Example 1. A spore suspension of YS-44442 or YS-45494 (9ml) stored in GL (10_glycerol_(—)5_lactose) at −80_ was opened inlaminar flower and added into a 3 L shake flask containing 600 ml of theseed medium as described in Example 1. The seed culture was incubated at27_ for 24 to 48 hrs in a rotary shaker at 200 to 220 rpm (growthphase). Afterward, 1.6 L of the seed culture was incubated into a 30 Lfermentor containing 16 L fermentation medium as described in Example 1in which 0.1 ml antifoam (LG-109) per liter was further added. Thefermentation was conducted at 27_with an agitation at 100 to 150 rpm andan air supply of 0.9 volume air per volume broth per minute for 24 to 48hrs. Afterward, compactin (50 to 60 g/L) was fed into the fermentationbroth in an amount of 10 to 30 ml/hr. After 2 to 3 days of feeding, thefermentation was finished. The fermentation broth was analysis by HPLCas described in Example 1, and the conversion efficiency of compactin topravastatin, the pravastatin yield and the epi-pravastatin ratio ofYS-44442 and YS-45494 are calculated as stated in Example 1 and shown inTable 9.

TABLE 9 Results of Pravastatin production by YS-44442 and YS-45494 withcontinuous feeding of compactin Pravastatin conversion Epi-pravastatinStrain yield efficiency ratio YS-44442 1.0 to 1.5 g/L 70 to 75% 6.5 to8% YS-45494 1.0 to 1.5 g/L 48 to 50% 1.8 to 3.0%

In comparison with the known microorganisms (e.g., Actinomadura sp. asdescribed in Peng, M. et al., J. Antibiotics, supra and Peng Y. and A.L. Demain, supra) which need a conversion period of 5 to 7 days (aftercompactin addition) to produce a yield of 0.326 to 0.821 g/L ofpravastatin with a compactin feeding concentration of 0.5 to 1.1 g/L,YS-44442 and YS-45494 of the invention are capable of producing a yieldof 1.0 to 1.5 g/L of pravastatin within 24 hours (after compactinaddition) with a compactin feeding concentration of 1.5 to 2.0 g/L. Inaddition, compactin can soon be added into a fermentation broth ofYS-44442 or YS-45494 after the fermentation broth is cultured for only18 hours (from inoculation with a seed culture). In the prior art,compactin cannot be added into a fermentation broth of Actinomadura sp.(Peng, M. et al., J. Antibiotics, supra and Peng Y. and A. L. Demain,supra) unless the broth is cultured for more than 2 days (frominoculation with a seed culture). Further, the pravastatin produced byYS-44442 or YS-45494 contains low ratio of epi-pravastatin (i.e.,6_-hydroxylation form of compactin) which does not have a desiredtherapeutic efficacy and even has a negative effect in clinicaltherapies. The two novel microorganisms of the invention exhibit ahigher tolerance to compactin and a better conversion efficiency ofcompactin to pravastatin.

Example 4

NTG Mutagenesis

A spore suspension of YS-44442 or YS-45494 was added into 10 ml of ISP2medium and incubated with shaking for 1 to 2 hours. Aftercentrifugation, the spore pellet was washed with 0.9% NaCl twice andsuspended in 3 ml of 0.9% NaCl. 2.5 ml of 0.2 M NaOH and 1 ml of 1000ppm NTG were added into the spore suspension, mixed well and incubatedat 27±1_for 10, 20, 30, 60 and 120 min. After incubation, the sporesuspension was added with 12% sodium thiosulfate and the up-layer aftercentrifugation was removed to eliminate the NTG. 10 ml of ISP2 mediumwas added into the spore suspension and incubated with shaking for 1 to2 hours. After centrifugation, the spore pellet was washed with 0.9%NaCl twice and suspended in 0.3 ml of 0.9% NaCl. After a 10-fold serialdilution, 0.1 ml of the diluted spore suspension was spread onto ISP2agar plates. After incubating at 27±1_ for 6 to 9 days, morphologies andthe numbers of the colonies appearing on the plates were observed andcounted, respectively. Preferably, the killing rate determined by thefollowing formula was between 50 to 99.5%.Killing rate=(numbers of cells without NTG treatment−numbers of cellsthrough NTG treatment)/numbers of cells without NTG treatment×100%

For example, if the agar plate spread by 10⁴ fold diluted sporesuspension through NTG treatment appears 28 colonies (equivalent to2.8×10⁶ cells) and the agar plate spread by 10⁶ fold diluted sporesuspension without NTG treatment appears 36 colonies (equivalent to3.6×10⁸ cells), then the killing rate is about 99.22%[100%×(360,000,000−2,800,000)/360,000,000]. Other mutagenesis usinganother mutagen can be conducted by the above method.

UV Irradiation Mutagenesis

A spore suspension of YS-44442 or YS-45494 was diluted by 10 fold serialdilution. 0.1 ml of diluted spore suspension was spread onto ISP2 agarplates. The agar plates were opened and treated with UV light for 30,60, 120, 240 and 480 seconds. After UV irradiation, the agar plates werecovered and immediately transferred into a black condition to avoidfurther light illumination. After incubating at 27±1_ for 6 to 9 days,morphologies and numbers of the colonies appearing on the plates wereobserved and counted. Preferably, the killing rate determined by thefollowing formula is between 99 to 99.999%.

Example 5 Bioactivity Assay of Mutants Derived from YS-44442 or YS-45494

After mutagenesis, the morphological variant was selected and put intoscrew tube with 6 to 10 glass beads and 0.5 ml deionized water, andmixed well. 0.1 to 0.15 ml of the suspension was refreshed in ISP2 slantagar and fermentated in a liquid medium. During fermentation, compactinwas added in a final concentration of 1.5 or 2.1 g/L and thebioconversion was proceeded at 27_for 24 to 48 hours. 0.2 ml of thefermentation broth was mixed with 1.8 ml deionized water. Aftercentriftigation, the supernatant was analyzed by HPLC as describedabove. Mutants derived from YS-44442 or YS-45494 were selected for theirpravastatin productivity, conversion efficiency and epi-pravastatinratio (see Table 10).

TABLE 10 Bioassay results of mutants derived from strains YS-44442 andYS-45494 Pravastatin conversion Epi-pravastatin Strain yield efficiencyratio 44442(parent) 1.08 g/L 72.0% 10.0%  3600-1(mutant 1.15 g/L 76.7%8.2% strain) 3600-2(mutant 1.30 g/L 86.7% 5.0% strain) * Compactin isfed in a final concentration of 1.5 g/L 45494(parent) 1.05 g/L 50.0%3.7% 8400-1(mutant 1.24 g/L 59.0% 3.0% strain) 8400-2(mutant 1.53 g/L72.9% 2.1% strain) * Compactin is fed in a final concentration of 2.1g/L

Example 6 Purification of Pravastatin by Ammonium Sulfate Precipitation

A pravastatin-containing fermentation broth (14L, pH 7.5 to 8.5) of themutant strain derived from the parent YS-44442 or YS-45494 was addedwith ammonium sulfate (4.5 to 7.0 kg) batchwise within 2 to 6 hours andthen stirred for 5 to 15 hrs at room temperature to give aprecipitation. The precipitation thus produced was separated byfiltration. The precipitation was dissolved in 100 to 200 ml of waterand stirred for 0.5 hrs to produce a solution. The solution was filteredthrough a bed of celite, washing the celite bed once with 100 ml ofwater. The filtrate was collected and the pH of the filtrate wasadjusted to pH 4.0 to 6.0 with 18% (w/v) HCl. The filtrate was thenmixed with 1.0 to 3.0 volume of ethyl acetate and stirred for 10 to 30min at room temperature. The emulsion was filtered by a bed of celite.After the phases were separated, the water phase was collected andextracted again with 1.0 to 3.0 volume of ethyl acetate. The ethylacetate phases obtained from such twice extractions were collectedtogether. The collected ethyl acetate phases were dried and decolorizedby adding with anhydrous magnesium (0.1 to 1.5% w/v) and activatedcarbon (0.1 to 1.5% w/v), stirring for 1 to 15 min at room temperature,filtering and washing with fresh ethyl acetate (100 ml), to yield94.495% (HPLC) of pravastatin.

To obtain a pravastatin sodium, the pravastatin purified as above wasdissolved in fresh ethyl acetate to produce a solution containingpravastatin in a concentration of 5.0 to 25.0 g/L. The solution wasadded with 0.2 to 5.0 M sodium-2-ethyl hexanoate in methanol/ethanol orisopropyl alcohol and stirred for 0.5 to 2.0 hrs at room temperature.The solution was then cooled to 0 to 20_, filtered and washed with freshethyl acetate, yielding 60 to 80% of pravastatin with a purity of 98.5%(HPLC) wherein the single impurity is in an amount of less than 0.3% andthe total impurity is less than 1.0%.

1. A process of isolating pravastatin, comprising the steps of (1)adding an ammonium sulfate into a first solution containing the(HMG)-CoA reductase inhibitor to produce a precipitation; (2) isolatingthe precipitation; (3) dissolving the precipitation with a polar solventto produce a second solution; (4) adjusting the pH of the secondsolution to about pH 4 to about PH6; and (5) extracting the secondsolution with an water immiscible solvent to isolate the (HMG)-CoAreductase inhibitor.
 2. The process of claim 1, wherein the (HMG)-CoAreductase inhibitor is selected from pravastatin, compactin andlovastatin.
 3. The process of claim 2, wherein the (HMG)-CoA reductaseinhibitor is pravastatin.
 4. The process of claim 1, wherein the firstsolution of Step (1) is a microbial fermentation broth.
 5. The processof claim 4, wherein the microbial fermentation broth is derived from amicroorganism capable of producing the (HMG)-CoA reductase inhibitor,said microorganism is selected form Streptomyces roseochrornogenus,Actinomadura, Aspergillus, Monascus, Penicillium, Paecilomyces,Hypomyces, Phoma, Pleurotus, Doratmyces, Eupenicillium, Gymnoaxus,Trichoderma, YS-44442 of Saccharothrix, YS-45494 of Saccharothrix, andthe mutants thereof.
 6. The process of claim 1, wherein the ammoniumsulfate of Step (1) is added into the first solution in an amount of 30to 60% (w/v) of the first solution.
 7. The process of claim 6, whereinthe ammonium sulfate is added to be saturated in the first solution. 8.The process of claim 1, wherein the water immiscible solvent of Step (5)is an organic solvent.
 9. The process of claim 8, wherein the organicsolvent is selected from ethyl acetate, acetone, toluene,dicholoromethane and isopropyl acetate.
 10. The process of claim 9,wherein the organic solvent is ethyl acetate.
 11. The process of claim1, further comprising a step of reacting the isolated (HMG)-CoAreductase inhibitor with an organic or inorganic cation source togenerate a salt form of the inhibitor.
 12. The process of claim 11,wherein the cation source is a sodium source.
 13. The process of claim12, wherein the sodium source is selected form NaOH, NA₂CO₃ sodiumacetate (anhydrous) and sodium-2-ethyl hexanoate.